当前期刊: Protein Expression and Purification Go to current issue    加入关注   
显示样式:        排序: 导出
我的关注
我的收藏
您暂时未登录!
登录
  • Obtaining active single chain antibody from bacterial inclusion bodies using metal ions
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-27
    Marzieh Najafi; Yaghoub Safdari

    Eukaryotic recombinant proteins expressed in bacterial cells usually aggregate within the cells as inclusion bodies. Despite the widely-accepted theory considering inclusion bodies as inactive materials, inclusion bodies may contain large amounts of correctly-folded active recombinant proteins. Molecules trapped in inclusion bodies can be released using a high pH solution (pH ≥ 11); however, they may undergo structural changes in such pH conditions that lead to their protein inactivation. Shifting in pH alongside the use of metal ions can help recover protein activity. The model protein we used in this study, 9R-Nimo.scFv, is highly active when extracted from bacterial inclusion bodies at high pH condition (pH 12), but loses its activity when pH is reduced to pH 7. We evaluated the capacity of nine salt solutions in terms of recovering protein activity in neutral pH conditions and found thatZnSO4 solution was the best one for this purpose. KNO3, and MnSO4 were also found to have a good capacity in recovering protein activity, as well.

    更新日期:2020-01-27
  • Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-27
    Walter Beata Maria; Szulc Aneta; Glinkowska Monika

    Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.

    更新日期:2020-01-27
  • A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-22
    Kavita Y. Hiremath; Narasimhappagari Jagadeesh; Shivakumar Belur; Supreeth S. Kulkarni; Shashikala R. Inamdar

    Background Lectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara were used to check the presence of lectin. Methods and results Here we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49 kDa and 17.4 kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Millibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29 cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and millibiose. LCL showed dose and time dependent growth inhibitory effects on HT29 cells with IC50 of 3.75 μg/ml at 48h. LCL has potent antibacterial and anti fungal activity. Conclusion LCL can be explored for its clinical potential.

    更新日期:2020-01-22
  • Recombinant β-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv libraries
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-22
    R.L. Anisimov; O.A. Ershova; A.V. Ershov; M.A. Filatova; S.A. Katorkin; V.M. Simonov

    Antibodies specific to β-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein β-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific binders were discovered and converted to full-size hIgG1 antibodies leading to highly stable binders with dissociation constants (Kd) in the range 10–40 nM. The antibodies were used further as ligands for affinity chromatography, where efficient and selective recovery of biologically active β-Glucocerebrosidase from cultured media of Chinese hamster ovary cells was demonstrated. β-Glucocerebrosidase was purified to nearly homogeneous state and had specific activity comparable to the commercially available preparations (40–44 U/mg protein). The obtained immunoaffinity sorbents have high capacity and can be easily regenerated.

    更新日期:2020-01-22
  • Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-21
    Mustafa Divyapicigil; Serife Seyda Pirincci Gokturk; Bengu Ergenoglu; Fatima Yucel; Esin Aslankaraoglu Akcael

    Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

    更新日期:2020-01-22
  • Enhancing soluble expression of sucrose phosphorylase in Escherichia coli by molecular chaperones
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-21
    Dong Yao; Jia Fan; Ruizhi Han; Jing Xiao; Qian Li; Guochao Xu; Jinjun Dong; Ye Ni

    Sucrose phosphorylase (SPase, EC 2.4.1.7) has a wide range of application in food, cosmetics, and pharmaceutical industries because of its broad substrate specificity. However, low SPase yields produced by wild-type strains cannot meet industrial requirements due to their complex metabolic regulation mechanisms. In this study, spase gene from Thermoanaerobacterium thermosaccharolyticum was cloned and expressed in Escherichia coli BL21 (DE3), leading to 7.05 U/mL (3.71 U/mg) of T. thermosaccharolyticum SPase (TtSPase) under optimum conditions. Co-expression of molecular chaperone teams pGro7 (GroES-GroEL), pG-KJE8 (DnaK-DnaJ-GrpE and GroES-GroEL), and pG-TF2 (GroES-GroEL-tig) significantly enhanced the TtSPase activities to 18.5 U/mg (59.2 U/mL), 9.52 U/mg (28.6 U/mL), and 25.7 U/mg (64.5 U/mL), respectively. Results suggested that GroES-GroEL chaperone combination could regulate protein folding processes and protect misfolded proteins from aggregation. The enzymatic characterization results showed that TtSPase had an optimal temperature of 60 °C and optimal pH of 6.5. In particular, it had high thermostability of T5030 = 67 °C and half-life (t1/2 at 70 °C) of 19 min. Furthermore, purified TtSPase was used for hydroquinone transglycosylation and 21% of molar production yield of α-arbutin was obtained. This study provides a TtSPase with high thermostability for potential industrial applications, and develops an effective strategy for improving soluble TtSPase production in E. coli.

    更新日期:2020-01-22
  • Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-15
    Uriel Rojas-Sánchez; Alberto Cristian López-Calleja; Blanca E. Millán-Chiu; Francisco Fernández; Achim M. Loske; Miguel A. Gómez-Lim

    Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L−1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

    更新日期:2020-01-15
  • Production of membrane proteins in industry: The example of GPCRs
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-14
    James C. Errey; Cédric Fiez-Vandal

    Whereas membrane proteins make up ∼23% of the human proteome, it is estimated that membrane proteins constitute more than 60% of current drug targets. With membrane proteins forming such a high percentage of drug targets relative to their abundance within the proteome, it is little wonder that drug companies need to rapidly access high quality membrane proteins for their drug discovery process. Newly devised technologies, such as rapid low-cost gene synthesis, novel detergents, and protein thermostabilisation strategies allow conventionally “undruggable” membrane proteins to be drugged. In this review, we survey the state-of-the-art gene design, expression and purification strategies, and protein thermostabilisation methods used within a modern drug discovery program, with a focus on G protein-coupled receptors.

    更新日期:2020-01-14
  • Heterologous production of porcine antimicrobial peptide PR-39 in Escherichia coli using SUMO and intein fusion systems
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-12
    Mandana Azari Samani; Sedigheh Asad; Mohammadreza Mehrnia

    About half a century after antibiotics discovery, multi-antibiotic-resistant bacteria posed a new challenge to medicine. Attempts to discover new antibiotics have drawn the attention to Antimicrobial Peptides (AMPs). The rapid growth, besides its known genetic and manipulation systems, makes E.coli the preferred host system for production of recombinant proteins on an industrial scale. To produce AMPs in E.coli, the application of fusion-tags with the aim of stability, solubility, and prevention of antimicrobial activity is one of the best practices in this regard. In this study, we presented two different expression systems for the production of PR-39 in E. coli; one in fusion with Intein-Chitin binding domain (CBD) and another in fusion with SUMO accompanied by polyhistidine affinity tag. Both were cloned in the NdeI-XhoI sites of pET-17b and transformed to E. coli BL21 (DE3) pLysS. Recombinant bacteria were cultured and induced with 0.4 mM IPTG at 30 °C. Expression and purification of target proteins were confirmed by Tricine- SDS-PAGE and dot blot analysis. Recovery of 250 μg PR-39/L from SUMO fusion system and 280 μg PR-39/L from the intein fusion system was achieved.

    更新日期:2020-01-13
  • Novel strategy for expression and characterization of rabies virus glycoprotein
    Protein Expres. Purif. (IF 1.291) Pub Date : 2020-01-03
    Rongqing Zhao; Yi Shan; Maohua Li; Zhiyong Lou; Ye Feng; Lisong Huang; Wenlin Ren; Panpan Wang; Yufei Sun; Ying Sun; Junchi Su; Hunter Sun; Dee Hong; Yu-hua Li; Rui-Feng Chen; Le Sun

    Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few μg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection.

    更新日期:2020-01-04
  • Expression, purification and characterization of 5′-nucleotidase from caterpillar fungus by efficient genome-mining
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-31
    Shan Lin; Cuibing Zhou; Hancheng Zhang; Zhiming Cai

    5′- nucleotidase (5′-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5′-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5′-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5′-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5′-NT exhibited excellent Km (1.107 mM), Vmax (0.113 μmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5′-nucleotidase. Moreover, substrate specificity analyses indicated that 5′-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5′-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

    更新日期:2019-12-31
  • Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-27
    Ya-Dong Ge; Lu-Lu Jiang; Shao-Lin Hou; Feng-Zhi Su; Peng Wang; Gen Zhang

    The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The Km values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 μM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu2+ and Zn2+. Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations.

    更新日期:2019-12-27
  • Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-26
    Brett Kinrade; Peter L. Davies; Tyler D.R. Vance

    Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ∼20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N- terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

    更新日期:2019-12-27
  • Refolding and purification of cGMP-grade recombinant human neurturin from Escherichia coli inclusion bodies
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-19
    Guoling Xi; Reza Esfandaria; Chester Bittencourt Sacramento; Hani Jouihan; Arun Sharma; Robert Roth; Thomas Linke

    Neurturin is a potent neurotrophic factor that has been investigated as a potential therapeutic agent for the treatment of neurodegenerative diseases, including Parkinson's disease, and, more recently, for the treatment of type II diabetes. However, purification of neurturin for clinical applications has been hampered by its low solubility in aqueous solutions. Here we describe the development of a scalable manufacturing process for recombinant neurturin from E. coli. inclusion bodies. Neurturin was refolded from solubilized inclusion bodies by fed-batch dilution refolding with a titer of 90 mg per liter refold and a refold yield of 89%. A two-step purification process using cation exchange and hydrophobic interaction chromatography, followed by formulation using tangential flow filtration resulted in an overall process yield of about 56 mg purified neurturin per liter refold. Solubility of neurturin during the purification process was maintained by the addition of 15% (w/v) glycerol to all buffers. For clinical applications and parenteral administration glycerol was replaced by 15% (w/v) sulfobutyl ether-beta-cyclodextrin (i.e. Captisol) in the drug substance formulation buffer. The final purified product had low or undetectable levels of product-related impurities and concentrations of process-related contaminants such as host cell proteins, host cell DNA, endotoxins and Triton X-100 were reduced more than 10,000-fold or below the limit of detection. Bioactivity of purified recombinant neurturin was demonstrated in a cell-based assay by activation of the MAPK signaling pathway.

    更新日期:2019-12-19
  • Cloning, expression and characterization of a thermo-alkali-stable xylanase from Aspergillus oryzae LC1 in Escherichia coli BL21(DE3)
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-12
    Nisha Bhardwaj; Vijay Kumar Verma; Venkatesh Chaturvedi; Pradeep Verma
    更新日期:2019-12-13
  • Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-04
    Xiaobing Peng, Guorui Peng, Xuni Li, Lifang Feng, Lingying Dong, Yuwen Jiang

    The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247–370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringens l-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

    更新日期:2019-12-04
  • Purification and biochemical characteristics of a novel fructosyltransferase with a high FOS transfructosylation activity from Aspergillus oryzae S719
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-12-02
    Susu Han, Tong Ye, Shuo Leng, Lixia Pan, Wei Zeng, Guiguang Chen, Zhiqun Liang

    Fructooligosaccharides (FOS) have widely used for the manufacture of low-calorie and functional foods, because they can inhibit intestinal pathogenic microorganism growth and increase the absorption of Ca2+ and Mg2+. In this study, the novel fructosyltransferase (FTase) from Aspergillus oryzae strain S719 was successfully purified and characterized. The specific activity of the final purified material was 4200 mg−1 with purification ratio of 66 times and yield of 26%. The molecular weight of FTase of A. oryzae S719 was around 95 kDa by SDS-PAGE, which was identified as a type of FTase by Mass Spectrometry (MS). The purified FTase had optimum temperature and pH of 55 °C and 6.0, respectively. The FTase showed to be stable with more than 80% of its original activity at room temperature after 12 h and maintaining activity above 90% at pH 4.0–11.0. The Km and kcat values of the FTase were 310 mmol L−1 and 2.0 × 103 min−1, respectively. The FTase was activated by 5 mmol L−1 Mg2+ and 10 mmol L−1 Na+ (relative activity of 116 and 114%, respectively), indicating that the enzyme was Mg2+ and Na+ dependent. About 64% of FOS was obtained by the purified FTase under 500 g L−1 sucrose within 4 h of reaction time, which was the shortest reaction time to be reported regarding the purified enzyme production of FOS. Together, these results indicated that the FTase of A. oryzae S719 is an excellent candidate for the industrial production of FOS.

    更新日期:2019-12-02
  • Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-29
    Tyler S. Beyett, Qiuyan Chen, Emily J. Labudde, Joseph Krampen, Prateek V. Sharma, John J.G. Tesmer

    G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids.

    更新日期:2019-11-30
  • Non-structural protein 1 from Japanese encephalitis virus expressed in E. coli retains its molecular weight and immunogenicity
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-29
    Jae-Won Choi, Hyo-Ji Eom, Hak Yong Kim

    Japanese encephalitis virus (JEV) is a member of the Flavivirus genus and has recently attracted attention as a high-risk pathogen in the Asia-Pacific region, with up to 30% mortality in the afflicted patients. Recent outbreaks of flavivirus-associated infections around the world have put the focus on non-structural protein 1 (NS1) as a candidate for diagnostic and vaccine researches on flaviviruses. Although the JEV NS1 protein has been expressed in eukaryotic cells, attempts to express JEV NS1 in E. coli are on due to advantages such as rapid growth, easy manipulation, low cost, and high yield. However, the challenges of low yield and poor solubility of the proteins expressed in E. coli remain to be overcome. Herein, we reported successful expression of the JEV NS1 protein in E. coli Rosetta(DE3) strain. We standardized the temperature, induction time, as well as the concentration of the inducer for optimizing the expression of JEV NS1 in E. coli. Further, we successfully obtained soluble JEV NS1 from inclusion bodies by partial refolding during elution and gradual refolding during dialysis. Furthermore, the JEV NS1 protein was found to retain its functional property and was able to induce an immune response in the mouse. Western blot and indirect enzyme-linked immunosorbent assay were performed using the blood of the immunized mouse and purified JEV NS1 in this study. Hence, JEV NS1 expressed in and isolated from E. coli Rosetta(DE3) strain holds potential for application in vaccine development and diagnostic studies to combat Japanese encephalitis outbreaks in the future.

    更新日期:2019-11-30
  • Expression and purification of codon-optimized cre recombinase in E. coli
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-27
    Srividya D, Anil H. Shyam Mohan, Saroja Narsing Rao

    The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and purified His6-Cre recombinase by immobilized metal affinity chromatography. N-terminal His6 tagged Cre recombinase obtained was approximately 26 fold purified and promoted the site-specific recombination of two loxP sites of linearized pLox2+ vector allowing the excision of a re-circularized plasmid and a short stretch of DNA containing the recombined loxP site. The results of the expression using two vectors, purification and activity assessment of His6 tagged Cre recombinase is presented here.

    更新日期:2019-11-28
  • Development of a biochemical and biophysical suite for integral membrane protein targets: A review
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-26
    Matthias Haffke, Myriam Duckely, Christian Bergsdorf, Veli-Pekka Jaakola, Binesh Shrestha

    The generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug design (SBDD) in drug discovery projects. We furthermore include timeline estimates and an overview of the target selection and biophysical screening approaches.

    更新日期:2019-11-27
  • Expression and purification of recombinant mouse CRISP4 using a baculovirus system
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-20
    Avinash S. Gaikwad, Khai Lee Loh, Anne E. O'Connor, Hugh H. Reid, Moira K. O'Bryan

    Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

    更新日期:2019-11-20
  • High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-20
    Xiaotong Lin, Liangbo Dong, Dou Yu, Bin Wang, Li Pan

    Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30–80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65–75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

    更新日期:2019-11-20
  • Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-19
    Chandler B. Est, Regina M. Murphy

    Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ∼15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.

    更新日期:2019-11-19
  • The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-19
    Navjit K. Paul, Karina A. Baksh, Joaquin F. Arias, Deborah B. Zamble

    Polyhistidine tags (His-tags) are commonly employed in protein purification strategies due to the high affinity and specificity for metal-NTA columns, the relative simplicity of such protocols, and the assumption that His-tags do not affect the native activities of proteins. However, there is a growing body of evidence that such tags can affect protein structure and function. In this study, we demonstrate that a His-tag impacts DNA complex formation by the C-terminal domain of the ??-subunit (??CTD) of Helicobacter pylori RNA polymerase in a metal-dependent fashion. The ??CTD was purified with a cleavable His-tag, and complex formation between αCTD, the nickel-responsive metalloregulator HpNikR, and DNA was investigated using electrophoretic mobility shift assays. An interaction between His-tagged ??CTD (His??CTD) and the HpNikR-DNA complex was observed; however, this interaction was not observed upon removal of the His-tag. Further analysis revealed that complex formation between HisαCTD and DNA is non-specific and dependent on the type of metal ions present. Overall, the results indicate that a histidine tag is able to modulate DNA-binding activity and suggests that the impact of metal affinity tags should be considered when analyzing the in vitro biomolecular interactions of metalloproteins.

    更新日期:2019-11-19
  • N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-15
    Balabhadra Khatiwada, Jeffrey A. Purslow, Eric S. Underbakke, Vincenzo Venditti

    Various fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partner and is promptly removed upon digestion with a protease. The presented protocol allows for the production of mg amounts of Alkbh5 and FTO in 1L of both rich and minimal media. We developed a liquid chromatography-mass spectrometry (LC-MS)-based assay to confirm that both proteins are enzymatically active. Furthermore, the LC-MS method developed here is applicable to other members of the AlkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases. The superior protein yield, afforded by our expression and purification method, will facilitate biochemical investigations into the biological function of the human RNA demethylases and endorse employment of EIN as a broadly applicable fusion tag for recombinant expression projects.

    更新日期:2019-11-15
  • New method for immobilising diverse proteins onto cubic micro-protein polyhedrin crystals
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-14
    Haruna Yuasa, Eiji Kotani, Hajime Mori, Keiko Takaki

    Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.

    更新日期:2019-11-14
  • Improving the heterologous expression of human β-defensin 2 (HBD2) using an experimental design
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-09
    Ligia Luz Corrales-García, Leobardo Serrano-Carreón, Gerardo Corzo

    At present, expressing antimicrobial peptides in bacterial models is considered a routine lab bench work. However, low expression yields of these types of proteins are usually obtained. In this work, the antimicrobial peptide human β-defensin 2 (HBD2) was obtained in low expression yields in Escherichia coli BL21(DE3). To improve the expression yields of HBD2, some variables such as cell density, temperature, and length of induction, as well as the inducer concentration, were investigated using a 24-factorial design of experiments (DoE). This approach allowed us to identify the identification of critical variables (main effects and interactions among factors) affecting bacterial HBD2 expression. After the evaluation of 19 different combination, the best condition to express HBD2 had a pre-induction temperature of 37 °C, a cell density of 1.0 U (600 nm), an induction temperature of 20 °C and a 0.1 mM of gene expression inducer (IPTG) over four hours. Under such conditions, the expression yield of the HBD2 peptide was one order of magnitude higher than the peptide expression performed initially.

    更新日期:2019-11-11
  • Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-08
    B.O. Araujo-Montoya, M.R. Senger, B.F. Gomes, G. Harris, R.J. Owens, F.P. Silva-Jr

    Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

    更新日期:2019-11-08
  • Recent developments of methyl-labeling strategies in Pichia pastoris for NMR spectroscopy
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-22
    Meng Zhang

    Nuclear magnetic resonance (NMR) spectroscopy is a primary structural biology method to characterize protein dynamics in solution. For large macromolecular systems, methyl-labeling in a perdeuterated background significantly improves the relaxation properties, while providing sensitive probes for structure and dynamics analysis. However, how to prepare methyl-labeled proteins, especially for functional eukaryotic proteins, remains to be a major bottleneck in this field. Due to its advantages in eukaryotic co-translational and post-translational modification, as well as high-density fermentation, Pichia pastoris has been a cost-effective platform strain for 13C, 15N-labeling and deuterium labeling since the early 2000's. Recently, some substantial progress has been made in methyl-labeling, such as the feasibility of 13C isoleucine δ1 methyl-labeling in perdeuterated background and the increased uptake of the Val/Leu precursor. Here, we systematically introduce the isotope-labeling strategies in P. pastoris, including strain engineering and detailed fermentation protocols in 13C, 15N-labeling and methyl-labeling, providing instructions and guidance for the future improvement of sample preparation for NMR spectroscopy.

    更新日期:2019-11-04
  • A bacterial endo-β-1,4-glucuronan lyase, CUL-I from Brevundimonas sp. SH203, belonging to a novel polysaccharide lyase family
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-09-20
    Masako Kikuchi, Naotake Konno, Tomohiro Suzuki, Yuta Fujii, Yutaka Kodama, Akira Isogai, Naoto Habu

    Cellouronate is a (1,4)-β-D-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose. We have previously isolated a cellouronate-degrading bacterial strain, Brevundimonas sp. SH203, that produces a cellouronate lyase (β-1,4-glucuronan lyase, CUL-I). In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized β-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family.

    更新日期:2019-11-04
  • Comparative analysis of fusion tags used to functionalize recombinant antibodies
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-09-26
    Gianluca Veggiani, Barbara Giabbai, Marta S. Semrau, Barbara Medagli, Vincenzo Riccio, Gregor Bajc, Paola Storici, Ario de Marco

    Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody. Furthermore, we assessed the efficiency of the derivatization process. The constructs always bore a 6xHis tag plus either the controls (EGFP and C-tag) or CLIP, HALO, AviTag, the LEPTG sequence recognized by Sortase A (Sortase tag), or a free cysteine. The advantages and drawbacks of the different systems were analyzed and discussed.

    更新日期:2019-11-04
  • The effect of N-glycosylation on the expression of the tetanus toxin fragment C in Pichia pastoris
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-09-21
    Nan Wang, Kevin Yueju Wang, Fangfang Xu, GangQiang Li, DeHu Liu

    The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a N-glycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.

    更新日期:2019-11-04
  • High yield recombinant expression and purification of oncogenic NSD1, NSD2, and NSD3 with human influenza hemagglutinin tag
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-09-26
    Yunpeng Shen, Masayo Morishita, Eric di Luccio

    The nuclear receptor-binding SET Domain (NSD) family consists of NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 histone methyltransferases that are crucial for chromatin remodeling. NSDs are implicated in developmental disorders such as Wolf-Hirschhorn and Sotos syndromes as well as various cancers including t(4; 14)(p16; q32) myeloma, an incurable cancer in plasma cells. NSDs have been the target of intensive study to understand their biological functions more fully and inform anti-cancer drug design. Recombinant protein expression and purification of human NSDs using an E. coli expression system are notoriously challenging, but the production of pure, stable, and active NSDs is essential for further studies. To overcome production challenges, we propose a cost-efficient approach optimized to produce a high yield of NSDs using a modified E. coli expression system. We found that tagging the NSDs with a human influenza hemagglutinin (HA) tag greatly improved the quality of the recombinant NSDs, resulting in more than 95% pure, stable, and active NSD-HAs, with an increase in production yield up to 22.4-fold and up to 6.25 mg/L from LB E. coli culture, and without further purification such as ion-exchange or size-exclusion chromatography.

    更新日期:2019-11-04
  • Identification and characterisation of a fluorinase from Actinopolyspora mzabensis
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-05
    Selisha Ann Sooklal, Charles De Koning, Dean Brady, Karl Rumbold

    The incorporation of fluorine has been shown to improve the biophysical and bioactive properties of several organic compounds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyse a C-F bond, hence, have vast potential to be applied as biocatalysts in the preparation of fine chemicals. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of soluble protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography, >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ± 0.03 μM min−1 mg−1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.

    更新日期:2019-11-04
  • A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-04
    Marina Y. Linova, Michael W. Risør, Sanne E. Jørgensen, Zohra Mansour, Jacob Kaya, Jens J. Sigurdarson, Jan J. Enghild, Henrik Karring

    The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403–621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416–621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying l-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M l-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

    更新日期:2019-11-04
  • Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-09
    Muhammad Umair Naseem, Nadeem Ahmed, Mohsin Ahmad Khan, Saad Tahir, Ahmad Usman Zafar

    Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.

    更新日期:2019-11-04
  • Cloning, expression, purification and biochemical characterization of recombinant metallothionein from the white shrimp Litopenaeus vannamei
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-15
    Jorge Duarte-Gutiérrez, Lilia Leyva-Carrillo, Miguel A. Martínez-Téllez, Rosa O. Méndez-Estrada, Monserrath Felix-Portillo, Gloria Yepiz-Plascencia

    Metallothioneins (MTs) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal intoxication and hypoxia (oxygen deficiency). During hypoxia the expression of the MT gene is induced in the shrimp Litopenaeus vannamei; however, the MT protein coded by this gene has not been purified nor characterized. In this work, the coding sequence of L. vannamei MT was cloned and overexpressed in Escherichia coli as a fusion protein, containing an intein and a chitin binding domain (CBD). The MT was purified by chitin affinity chromatography and its antioxidant capacity and ability to bind cadmium (Cd) and copper (Cu) were evaluated. This MT has an antioxidant capacity of 27.23 μM equivalent to Trolox in a 100 μg/mL solution. Addition of CdCl2 to the culture media augments 273-fold the Cd content, while addition of CuCl2 increases Cu content 569-fold in the purified MT. Thus, the shrimp MT gene codes for a functional protein that has antioxidant capacity and binds Cu and Cd.

    更新日期:2019-11-04
  • Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-16
    Sedigheh Mohammadzadeh, Fatemeh Moradian, Sakineh Yeganeh, Bahram Falahatkar, Sylvain Milla

    GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

    更新日期:2019-11-04
  • Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-18
    Yu Liu, Yanling Li, Sheng Tong, Min Yuan, Xiaoyun Wang, Junyao Wang, Yanhua Fan

    Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 μg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2–8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.

    更新日期:2019-11-04
  • Screening and production of an affibody inhibiting the interaction of the PD-1/PD-L1 immune checkpoint
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-20
    Lei Jing, Juanjuan Liu, Dongxu Cui, Yuyin Li, Zhenxing Liu, Li Tao, Qing Zhao, Aipo Diao

    An affibody is a 58 amino acids peptide derived from the Z domain of staphylococcal protein A and generally applied in areas such as imaging diagnosis, clinical therapeutics and biotechnology research. To screen for an affibody targeting the immune checkpoint PD-L1, a combinatorial affibody library was generated in yeast using degenerate overlap PCR primers and In-fusion technology. Z-j1 and Z-j2 affibodies targeting the Ig-like V domain of PD-L1 were screened and identified from this combinatorial library using the yeast two hybrid system. The Z-j1 and Z-j2 recombinant affibody proteins were over produced in E.coli and purified. ELISA and GST pull-down assays showed that recombinant Z-j1 and Z-j2 affibody proteins bound with high affinity to PD-L1 and inhibited the interaction of PD-1/PD-L1. Thus, novel affibodies targeting the immune checkpoint PD-1/PD-L1 were identified and produced in this study and have the potential to be used in cancer immunotherapy.

    更新日期:2019-11-04
  • Strategies for successful isolation of a eukaryotic transporter
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-23
    Savvas Saouros, Cristina Cecchetti, Alex Jones, Alexander D. Cameron, Bernadette Byrne

    The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643–672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3Δ1-642 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3Δ1-642 than DDM. Isolation of OsBOR3Δ1-642 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3Δ1-642 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3Δ1-642 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.

    更新日期:2019-11-04
  • Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-04
    Minh Tan Nguyen, Yunseok Heo, Bich Hang Do, Sangki Baek, Chong Jai Kim, Yeon Jin Jang, Weontae Lee, Han Choe

    Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.

    更新日期:2019-11-04
  • Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-29
    Muhammad Arif, Qamar Bashir, Masood Ahmad Siddiqui, Naeem Rashid

    Phosphoribosyl anthranilate isomerase is involved in the isomerization of phosphoribosyl anthranilate to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. In the present study, trpFGt, a gene encoding phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis, was cloned and expressed in Escherichia coli. The gene product, TrpFGt, was produced in E. coli in soluble and active form. Molecular characterization revealed that recombinant TrpFGt was highly efficient and stable. The apparent Vmax and Km values were 480 μmol min−1 mg−1 and 1.15 μM, respectively. The half-life of the enzyme was 90 min at 60 °C. Apart from thermostability, TrpFGt was highly stable against protein denaturants such as urea. There was no significant change in activity even after treatment with 8 M urea. To the best of our knowledge, TrpFGt, is the most active and stable phosphoribosyl anthranilate isomerase characterized to date and this is the first characterization of TrpF from the genus Geobacillus.

    更新日期:2019-11-04
  • Removal of half antibody, hole-hole homodimer and aggregates during bispecific antibody purification using MMC ImpRes mixed-mode chromatography
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-04
    Jiaqin Tang, Xudong Zhang, Tao Chen, Ying Wang, Yifeng Li

    During recombinant production of asymmetric IgG-like bispecific antibodies (bsAbs), various by-products are often observed due to unbalanced chain expression and incorrect chain pairing. Among them, half antibody and homodimer are found with high frequency. In this work, with a case study we demonstrated that Capto MMC ImpRes mixed-mode chromatography can effectively remove these two by-products as well as antibody aggregates under optimized conditions. This makes MMC ImpRes a powerful tool for bsAb purification.

    更新日期:2019-11-04
  • Surface display of classical swine fever virus E2 glycoprotein on gram-positive enhancer matrix (GEM) particles via the SpyTag/SpyCatcher system
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-02
    Ding Li, Haoming Zhang, Li Yang, Jin Chen, Yuanpeng Zhang, Xiaoming Yu, Qisheng Zheng, Jibo Hou

    The E2 envelope protein is the main protective antigen of classical swine fever virus (CSFV). Importantly, gram-positive enhancer matrix (GEM) particles can work as an immunostimulant and/or carrier system to improve the immune effect of antigens. In this study, the artificially designed E2-Spy was expressed and glycosylated in Pichia pastoris, and subsequently conjugated with SpyCatcher-PA which was expressed in Escherichia coli. The conjugated E2-Spy-PA was displayed on the surface of GEM particles, generating the E2-Spy-PA-GEM complex. Blocking ELISA analysis and neutralization assays showed that both E2-Spy and E2-Spy-PA-GEM complexes induced high levels of anti-CSFV antibodies in mice. Furthermore, statistical analyses indicated that the E2-Spy-PA-GEM complex exhibited enhanced immunogenicity compared with E2-Spy alone.

    更新日期:2019-11-04
  • A high yielding IFNAR1 ECD mammalian expression process for use in autoimmune disease drug development
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-02
    Caroline Kittinger, Arnita Barnes, Alan Hunter, LeeAnn Machiesky, Sandrina Phipps, Anthony Shannon, Robert Stadelman, Susan Wilson, Ellen O'Connor

    Interferon-alpha receptor 1 (IFNAR1) is a target of interest for recombinant biotherapeutics that block the JAK/STAT pathway. This pathway is believed to play a role in many diseases including Hepatitis B and C, Herpes Simplex, Multiple Sclerosis, and other autoimmune disorders. By using IFNAR1 as a target to block Type I IFN from binding to the JAK/STAT pathway and prevent activation of this target, autoimmune disease progression can be modulated. Current IFNAR1 extracellular domain (ECD) expression and purification protocols are labor intensive with low product yield and limited scalability. In this work, we evaluate three different expression systems (baculovirus, human embryonic kidney 293 (HEK293×), and Chinese hamster ovary (CHO)) to improve expression of IFNAR1 ECD. We demonstrate the benefits of utilizing mammalian CHO cell transient transfection to increase expression titer, as well as an improved two-step purification process performed using immobilized metal affinity chromatography (IMAC) as the capture step and Ceramic Hydroxyapatite (CHT) Type II for HMW impurity removal in flow through mode. This process showed an 20-fold increase in productivity compared to the baseline process as measured by grams purified per liter of cell culture fluid. Lastly, the improved process showed good scalability, enabling efficient purification of 3.6 g of product from a 30 L scale bioreactor.

    更新日期:2019-11-04
  • Expression, purification, and structural analysis of the full-length human integral membrane protein γ-sarcoglycan
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-01
    Michael Jamaladdine, Michael S. Harris, Leshani Liyanage, Gabriel A. Cook

    Mutation of the gene encoding γ-sarcoglycan (SGCG), an integral membrane protein responsible for maintaining the integrity of the muscle cell sarcolemma, results in Limb-Girdle Muscular Dystrophy (LGMD), a congenital disease with no current treatment options. This member of the sarcoglycan glycoprotein family is a vital component of the Dystrophin Complex, which together facilitate normal muscle function. However, very little is known about the structure and dynamics of these proteins, and of membrane glycoproteins in general. This is due to a number of factors, including their complexity, heterogeneity and highly-specific native environments. The expression, purification, and structural study of membrane proteins is further impeded by their hydrophobic nature and consequent propensity to aggregate in aqueous solutions. Here, we report the first successful expression and purification of milligram quantities of full-length recombinant SGCG, utilizing fusion protein-guided overexpression to inclusion bodies in Escherichia coli. Purification of SGCG from the fusion protein, TrpΔLE, was facilitated using chemical cleavage. Cleavage products were then isolated by size-exclusion chromatography. Successful purification of the protein was confirmed using SDS-PAGE and mass spectroscopy. Finally, solution nuclear magnetic resonance spectroscopy of uniformly 15N-labeled SGCG in detergent environments was performed, yielding the first spectra of the full-length membrane glycoprotein, SGCG. These results represent the initial structural studies of SGCG, laying the foundation for further investigation on the interaction and dynamics of other integral membrane proteins. More specifically, these data allow for opportunities in the future for enhanced treatment modalities and cures for LGMD.

    更新日期:2019-11-04
  • Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-11-01
    Ding Li, Junchen Wu, Jin Chen, Dong Zhang, Yuanpeng Zhang, Xuwen Qiao, Xiaoming Yu, Qisheng Zheng, Jibo Hou

    Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.

    更新日期:2019-11-04
  • Expression and purification of recombinant G protein-coupled receptors: A review
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-31
    Daniel N. Wiseman, Abigail Otchere, Jaimin H. Patel, Romez Uddin, Naomi L. Pollock, Sarah J. Routledge, Alice J. Rothnie, Cathy Slack, David R. Poyner, Roslyn M. Bill, Alan D. Goddard

    Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

    更新日期:2019-11-04
  • Cloning, characterization and expression analysis of glutathione S-transferase from the Antarctic yeast Rhodotorula mucilaginosa AN5
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-10-25
    Cuijuan Shi, Xiaofei Wang, Zijie Xiao, Ruiqi Wang, Yongping Qiao, Guangfeng Kan

    The gene for glutathione S-transferase (GST) in Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 was cloned and expressed in Escherichia coli and named RmGST. Sequence analysis showed that the RmGST gene contained a 843 bp open reading frame, which encoded 280 amino acid residues with a calculated molecular mass of 30.4 kDa and isoelectric point of 5.40. RmGST has the typical C- and N-terminal double domains of glutathione S-transferase. Recombinant RmGST (rRmGST) was expressed in E. coli to produce heterologous protein that had a high specific activity of 60.2 U/mg after purification. The apparent Km values of rRmGST for glutathione and 1-chloro-2,4-dinitrobenzene were 0.35 mM and 0.40 mM, respectively. Optimum enzyme activity was measured at 35 °C and at pH 7.0 and complete inactivation was observed after incubation at 55 °C for 60 min rRmGST tolerated high salt concentrations (1.0 M NaCl) and was stable at pH 3.0. Additionally, the recombinant protein nearly kept whole activity in Hg2+ and Mn2+, and could tolerate Ca2+, Cu2+, Mg2+, Cd2+, EDTA, thiourea, urea, Tween-80, H2O2 and Triton X-100. Real-time quantitative PCR showed that relative expression of the GST gene was significantly increased under Cu2+ and low temperature stress. These results indicate that rRmGST is a typical low thermostable enzyme, while its other characteristics, heavy metal and low temperature tolerance, might be related to its Antarctic home environment.

    更新日期:2019-11-04
  • Functional Expression and Purification of CYP93C20,a Plant Membrane-Associated Cytochrome P450 from Medicago truncatula
    Protein Expres. Purif. (IF 1.291) Pub Date : 2010-12-04
    Zhenzhan Chang, Xiaoqiang Wang, Risheng Wei, Zhouying Liu, Hong Shan, Guizhen Fan, Hongli Hu

    Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni2+-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.

    更新日期:2019-11-04
  • A multi-column plate adapter provides an economical and versatile high-throughput protein purification system.
    Protein Expres. Purif. (IF 1.291) Pub Date : 2018-07-25
    Matthew J Dominguez,Benjamin J Lantz,Rebecca J Rhode,Zoey L Sharp,Krysten C Finney,Valeria Jaramillo Martinez,Elliott J Stollar

    Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

    更新日期:2019-11-01
  • Expression, purification, and in vitro cysteine-10 modification of native sequence recombinant human transthyretin.
    Protein Expres. Purif. (IF 1.291) Pub Date : 2007-02-24
    Jonathan S Kingsbury,Elena S Klimtchuk,Roger Théberge,Catherine E Costello,Lawreen H Connors

    Transthyretin (TTR) is a serum protein that is also a prominent component of deposits in two different types of systemic amyloid disease, senile systemic and familial TTR amyloidoses. Studies of recombinant TTR (rTTR) have provided many insights into the relationship between protein structure and amyloidogenicity. Yet, there is no existing recombinant system that results in high yield production of a protein that is identical in primary structure to human TTR. To date, most published studies have generated rTTR using the human gene sequence, which is poorly expressed in Escherichia coli. In addition, the gene sequence has been flanked by a 3' AUG start codon to initiate translation, resulting in the expression of a protein containing an N-terminal methionine residue not present in the human protein. We present an improved technique which can be used to generate large quantities of human native sequence TTR. Our recombinant system utilizes a gene containing codons altered for efficient expression in E. coli and an N-terminal polyhistidine tag for simplified purification. Optimization of this system was accomplished by generating a modified polyhistidine tag that was efficiently removed by dipeptidyl aminopeptidase I (DAPase). This is the first report detailing an effective and useful method for producing rTTR containing an amino acid sequence identical to human TTR. Furthermore, we describe the thiol modification of the recombinant protein to achieve exact replication of the several prominent post-translationally modified forms of TTR that have been identified in human serum.

    更新日期:2019-11-01
  • A method for improving protein A chromatography's aggregate removal capability.
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-03-05
    Yuan Zhang,Ying Wang,Yifeng Li

    Protein A chromatography is generally less effective at removing antibody aggregates under typical conditions. We recently developed a method that can significantly improve Protein A's aggregate removal capability. This method involves adding calcium chloride/polyethylene glycol (PEG) or sodium chloride/PEG combination to wash and elution buffers. Each salt alone showed some resolution-enhancing effect when its concentration reaches a certain level, and this effect was significantly enhanced by the presence of PEG, which itself had no effect on resolution. The synergistic effect of salt and PEG results in almost complete separation of monomer from aggregates. For the particular case used for method development and demonstration, in comparison with the control run the optimized procedure reduces aggregates in elution pool from 20% to 3-4%. This new method, by facilitating aggregate removal at the capture step, improves the overall robustness of downstream process.

    更新日期:2019-11-01
  • Purification and characterization of the extracellular region of human receptor tyrosine kinase like orphan receptor 2 (ROR2).
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-03-04
    Yuan Li,Xu Han,Wenqing Xu,Zihe Rao,Xin Li

    Receptor tyrosine kinase like orphan receptor 2 (ROR2) is a co-receptor for some Wnt proteins including Wnt5a that activate the noncanonical Wnt/planar cell polarity (PCP) signaling pathway. Upregulation of ROR2 is associated with several cancer forms. The extracellular region of ROR2, which contains an immunoglobulin (Ig)-like domain, a Frizzled like cysteine-rich domain (CRD) and a Kringle domain, is a potential anticancer drug target. The structural and biochemical properties of the ROR2 extracellular region remain largely unexplored. Here we describe the mapping and purification, using a baculovirus - insect cell system, of a near-full-length ROR2 extracellular fragment (residues 53-402), which is well-behaved and suitable for future structural and biochemical analysis. We show that the extracellular region of ROR2 per se is monomeric in solution. Different monoclonal antibodies raised against the purified ROR2 protein can specifically recognize the protein and can either inhibit or activate the PCP activity in a cell-based assay, and are thus potentially useful for future mechanistic and therapeutic/diagnostic studies. The biological relevance of these antibodies further demonstrates that the purified recombinant ROR2 protein is properly folded and biochemically active.

    更新日期:2019-11-01
  • The adequate amount of sodium chloride in Protein A wash buffer for effective host cell protein clearance.
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-03-03
    Jinyu Han,Ju Yang,Ying Wang,Yifeng Li

    Post-load column wash in Protein A chromatography can effectively improve host cell protein (HCP) clearance. A commonly used wash additive for this purpose is sodium chloride. However, the adequate amount of sodium chloride required for effective HCP clearance is less consistent in literature. In this study we investigated the impact of different amounts of sodium chloride on HCP clearance with five monoclonal antibodies (mAbs). For each mAb, elution pool HCP levels from runs under different wash conditions are compared. For all five mAbs, the data suggested that 250 mM would be an adequate amount for the salt to largely achieve its HCP reducing effect. The same conclusion is also reached for calcium chloride, a less commonly used but equally effective Protein A wash additive for HCP clearance.

    更新日期:2019-11-01
  • Production of recombinant human acid β-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-03-02
    Jae-Wan Jung,Hong-Yeol Choi,Nguyen-Xuan Huy,Heajin Park,Ha Hyung Kim,Moon-Sik Yang,Seung-Hoon Kang,Dong-Il Kim,Nan-Sun Kim

    Gaucher disease is an inherited metabolic disease caused by genetic acid β -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

    更新日期:2019-11-01
  • Cloning and high-level SUMO-mediated fusion expression of a serine protease inhibitor from Hyphantria cunea Drury that exhibits activity against papain.
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-02-27
    Ming Sang,Chen Xu,Zhiheng Wei,Xiaolong Wu,Yuxing Guo,Jianfeng Li,Zhiguo Wang,Jiaxin Zhang

    Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities. Todate some functional roles for serpins in Hyphantria cunea Drury have been identified. Here, new functional features of the H. cunea serine protease inhibitor (dHC-serpin) were characterized. In this study, the cDNA encoding serpin was amplified from H. cunea (dHC) pupa fat body total RNA using RT-PCR. The full-length dHC-serpin cDNA encoded a protein of 440 amino acids with a predicted 19-amino acid signal peptide and a 421-amino acid functional domain. The functional domain was cloned into a pSUMO vector and transformed into Escherichia coli, resulting in the production of a pSUMO-dHC-serpin fusion protein. The soluble form of this protein was then purified by Ni-IDA chromatography. The SUMO-dHC-serpin fusion protein was then cleaved using a SUMO protease and purified again by Ni-IDA chromatography. dHC-serpin did not inhibit trypsin, elastase, proteinase K or cathepsin B, but strongly inhibited papain. The inhibitor retained its inhibitory activity over a broad range of pH (pH 2-12), temperature (20-50 °C), and DTT concentration (up to 100 mM). A complete loss of inhibitory activity was observed at pH 13 and 70 °C. Serpins generally serve as inhibitors that use a mobile reactive center loop (RCL) as bait to trap protease targets. dHC-serpin, like others serpins, binds papain using the RCL structure.

    更新日期:2019-11-01
  • Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II).
    Protein Expres. Purif. (IF 1.291) Pub Date : 2019-02-19
    Keshav V,Achilonu I,Dirr Hw,Kondiah K

    PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, β-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

    更新日期:2019-11-01
Contents have been reproduced by permission of the publishers.
导出
全部期刊列表>>
2020新春特辑
限时免费阅读临床医学内容
ACS材料视界
科学报告最新纳米科学与技术研究
清华大学化学系段昊泓
自然科研论文编辑服务
加州大学洛杉矶分校
上海纽约大学William Glover
南开大学化学院周其林
课题组网站
X-MOL
北京大学分子工程苏南研究院
华东师范大学分子机器及功能材料
中山大学化学工程与技术学院
试剂库存
天合科研
down
wechat
bug